Identification of New Compounds against PRRSV Infection by Directly Targeting CD163.

The porcine reproductive and respiratory syndrome viruses (PRRSV) led to a global panzootic and huge economical losses to the pork industry. PRRSV targets the scavenger receptor CD163 for productive infection. However, currently no effective treatment is available to control the spread of this disease. Using bimolecular fluorescence complementation (BiFC) assays, we screened a set of small molecules potentially targeting the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163. We found that the assay examining protein-protein interactions (PPI) between PRRSV glycoprotein 4 (GP4) and the CD163-SRCR5 domain mainly identifies compounds that potently inhibit PRRSV infection, while examining the PPI between PRRSV-GP2a and the SRCR5 domain maximized the identification of positive compounds, including additional ones with various antiviral capabilities. These positive compounds significantly inhibited both types 1 and 2 PRRSV infection of porcine alveolar macrophages. We confirmed that the highly active compounds physically bind to the CD163-SRCR5 protein, with dissociation constant (KD) values ranging from 28 to 39 µM. Structure-activity-relationship (SAR) analysis revealed that although both the 3-(morpholinosulfonyl)anilino and benzenesulfonamide moieties in these compounds are critical for the potency to inhibit PRRSV infection, the morpholinosulfonyl group can be replaced by chlorine substituents without significant loss of antiviral potency. Our study established a system for throughput screening of natural or synthetic compounds highly effective on blocking of PRRSV infection and shed light on further SAR modification of these compounds. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Current vaccines cannot provide cross protection against different strains, and there are no effective treatments available to hamper the spread of this disease. In this study, we identified a group of new small molecules that can inhibit the PRRSV interaction with its specific receptor CD163 and dramatically block the infection of both types 1 and type 2 PRRSVs to host cells. We also demonstrated the physical association of these compounds with the SRCR5 domain of CD163. In addition, molecular docking and structure-activity relationship analyses provided new insights for the CD163/PRRSV glycoprotein interaction and further improvement of these compounds against PRRSV infection.

Small molecules block the interaction between porcine reproductive and respiratory syndrome virus and CD163 receptor and the infection of pig cells.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the pork industry globally. PRRS is caused by PRRS virus (PRRSV). Currently there are no effective treatments against this swine disease. METHODS: Through artificial intelligence molecular screening, we obtained a set of small molecule compounds predicted to target the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163, which is a cell surface receptor specific for PRRSV infection. These compounds were screened using a cell-based bimolecular fluorescence complementation (BiFC) assay, and the function of positive hit was further evaluated and validated by PRRSV-infection assay using porcine alveolar macrophages (PAMs). RESULTS: Using the BiFC assay, we identified one compound with previously unverified function, 4-Fluoro-2-methyl-N-[3-(3-morpholin-4-ylsulfonylanilino)quinoxalin-2-yl]benzenesulfonamide (designated here as B7), that significantly inhibits the interaction between the PRRSV glycoprotein (GP2a or GP4) and the CD163-SRCR5 domain. We further demonstrated that compound B7 inhibits PRRSV infection of PAMs, the primary target of PRRSV in a dose-dependent manner. B7 significantly inhibited the infection caused by both type I and type II PRRSV strains. Further comparison and functional evaluation of chemical compounds structurally related to B7 revealed that the 3-(morpholinosulfonyl)aniline moiety of B7 or the 3-(piperidinylsulfonyl)aniline moiety in a B7 analogue is important for the inhibitory function against PRRSV infection. CONCLUSIONS: Our study identified a novel strategy to potentially prevent PRRSV infection in pigs by blocking the PRRSV-CD163 interaction with small molecules.

Cryptotanshinone protects porcine alveolar macrophages from infection with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV) infection, imposes enormous economic impact to the world pork industry. Currently there is no effective treatment to prevent PRRSV infection in swine. We report that the natural compound cryptotanshinone (Cpt) effectively inhibits the infection of various strains of PRRSV to porcine alveolar macrophages (PAMs), the primary cell target of PRRSV in vivo. Mechanistically, Cpt inhibits the activation of signal transducer and activator of transcription 3 (STAT3), and blocks the interleukin 10 (IL-10) stimulated as well as the basal level CD163 expression in PAMs. Cpt-treatment of PAMs is effective when applied either before or after PRRSV infection, with the combined pre- and post-PRRSV infection treatment resulting in the most significant, dose-dependent inhibition of PRRSV infection. Cpt inhibited both type I/II PRRSV infection in PAMs. Our study identified a new approach to prevent/treat PRRSV infection of pigs with natural compounds.

Broad Protection of Pigs against Heterologous PRRSV Strains by a GP5-Mosaic DNA Vaccine Prime/GP5-Mosaic rVaccinia (VACV) Vaccine Boost.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) viruses are a major cause of disease and economic loss in pigs worldwide. High genetic diversity among PRRSV strains is problematic for successful disease control by vaccination. Mosaic DNA and vaccinia (VACV) vaccines were developed in order to improve protection against heterologous PRRSV strains. METHODS: Piglets were primed and boosted with GP5-Mosaic DNA vaccine and recombinant GP5-Mosaic VACV (rGP5-Mosaic VACV), respectively. Pigs vaccinated with rGP5-WT (VR2332) DNA and rGP5-WT VACV, or empty vector DNA and empty VACV respectively, served as controls. Virus challenge was given to separate groups of vaccinated pigs with VR2332 or MN184C. Necropsies were performed 14 days after challenge. RESULTS: Vaccination with the GP5-Mosaic-based vaccines resulted in cellular reactivity and higher levels of neutralizing antibodies to both VR2332 and MN184C PRRSV strains. In contrast, vaccination of animals with the GP5-WT vaccines induced responses only to VR2332. Furthermore, vaccination with the GP5-Mosaic based vaccines resulted in protection against challenge with two heterologous virus strains, as demonstrated by the significantly lower viral loads in serum, tissues, porcine alveolar macrophages (PAMs), and bronchoalveolar lavage (BAL) fluids, and less severe lung lesions after challenge with either MN184C or VR2332, which have only 85% identity. In contrast, significant protection by the GP5-WT based vaccines was only achieved against the VR2332 strain. Conclusions: GP5-Mosaic vaccines, using a DNA-prime/VACV boost regimen, conferred protection in pigs against heterologous viruses.

Canine circoviral hemorrhagic enteritis in a dog in Connecticut.

A 5-mo-old Bassett Hound-Labrador Retriever cross was autopsied following a bout of lethargy, inappetence, and bleeding gums. Mucous membranes were white, and the small intestine was blue-black; the colon contained black feces. The spleen was swollen, and multiple lymph nodes were enlarged and hemorrhagic. Microscopically, the small intestine had focal crypt cell necrosis and circumferential transmural vasculitis, the latter the cause of infarction and the blue-black coloration. Lymphocytes were necrotic in spleen and lymph nodes, and erythrophagocytosis was present in some nodes. Vasculitis was present in brain, meninges, lung, liver, and kidneys. Electron microscopy revealed aggregates of 15-18 nm round viral particles in damaged crypt cells and in the endothelium of small blood vessels. Electron-dense intracytoplasmic inclusions consisting of paracrystalline-arrayed virus were demonstrated in macrophages in medullary lymph node sinuses. These virions were identified as circovirus, which was confirmed by real-time PCR and sequencing.

Correction: A PRRSV GP5-Mosaic vaccine: Protection of pigs from challenge and ex vivo detection of IFNγ responses against several genotype 2 strains.

[This corrects the article DOI: 10.1371/journal.pone.0208801.].

A PRRSV GP5-Mosaic vaccine: Protection of pigs from challenge and ex vivo detection of IFNγ responses against several genotype 2 strains.

Porcine reproductive and respiratory syndrome virus (PRRSV), is a highly mutable RNA virus that affects swine worldwide and its control is very challenging due to its formidable heterogeneity in the field. In the present study, DNA vaccines constructed with PRRSV GP5-Mosaic sequences were complexed to cationic liposomes and administered to experimental pigs by intradermal and intramuscular injection, followed by three boosters 14, 28 and 42 days later. The GP5-Mosaic vaccine thus formulated was immunogenic and induced protection from challenge in vaccinated pigs comparable to that induced by a wild type (VR2332) GP5 DNA vaccine (GP5-WT). Periodic sampling of blood and testing of vaccine-induced responses followed. Interferon-γ (IFN-γ) mRNA expression by virus-stimulated peripheral blood mononuclear cells (PBMCs) of GP5-Mosaic-vaccinated pigs was significantly higher compared to pigs vaccinated with either GP5-WT or empty vector at 21, 35 and 48 days after vaccination. Cross-reactive cellular responses were also demonstrated in GP5-Mosaic vaccinated pigs after stimulation of PBMCs with divergent strains of PRRSV. Thus, significantly higher levels of IFN-γ mRNA were detected when PBMCs from GP5-Mosaic-vaccinated pigs were stimulated by four Genotype 2 strains (VR2332, NADC9, NADC30 and SDSU73), which have at least 10% difference in GP5 amino acid sequences, while such responses were recorded only upon VR2332 stimulation in GP5-WT-vaccinated pigs. In addition, the levels of virus-specific neutralizing antibodies were higher in GP5-Mosaic or GP5-WT vaccinated pigs than those in vector-control pigs. The experimental pigs vaccinated with either the GP5-Mosaic vaccine or the GP5-WT vaccine were partially protected from challenge with VR2332, as measured by significantly lower viral loads in sera and tissues and lower lung lesion scores than the vector control group. These data demonstrate that the GP5-Mosaic vaccine can induce cross-reactive cellular responses to diverse strains, neutralizing antibodies, and protection in pigs.

A self-adjuvanted nanoparticle based vaccine against infectious bronchitis virus.

Infectious bronchitis virus (IBV) affects poultry respiratory, renal and reproductive systems. Currently the efficacy of available live attenuated or killed vaccines against IBV has been challenged. We designed a novel IBV vaccine alternative using a highly innovative platform called Self-Assembling Protein Nanoparticle (SAPN). In this vaccine, B cell epitopes derived from the second heptad repeat (HR2) region of IBV spike proteins were repetitively presented in its native trimeric conformation. In addition, flagellin was co-displayed in the SAPN to achieve a self-adjuvanted effect. Three groups of chickens were immunized at four weeks of age with the vaccine prototype, IBV-Flagellin-SAPN, a negative-control construct Flagellin-SAPN or a buffer control. The immunized chickens were challenged with 5x10(4.7) EID50 IBV M41 strain. High antibody responses were detected in chickens immunized with IBV-Flagellin-SAPN. In ex vivo proliferation tests, peripheral mononuclear cells (PBMCs) derived from IBV-Flagellin-SAPN immunized chickens had a significantly higher stimulation index than that of PBMCs from chickens receiving Flagellin-SAPN. Chickens immunized with IBV-Flagellin-SAPN had a significant reduction of tracheal virus shedding and lesser tracheal lesion scores than did negative control chickens. The data demonstrated that the IBV-Flagellin-SAPN holds promise as a vaccine for IBV.

Search for evidence of recurring or persistent viruses in Crohn's disease.

New-onset Crohn's disease and acute flares are often associated with viral infections. The aim of this study was to search for evidence of persistent or recurrent viruses in patients. Tissue blocks were obtained from surgical specimens from patients and a control population. 111 samples were tested by PCR or RT-PCR, for EBV, CMV, HSV 1, HSV 2, HHV 8, pestiviruses, and enteroviruses. Additionally, seven sets of serum samples, including pre-operative and post-operative samples, from CD patients were analyzed serologically for antibodies to EBV. The tests revealed evidence of EBV nucleic acid in tissues of 11 patients from a total of 70 tested (15.7%) and in tissues of 3 of 41 control subjects (7.3%). Evidence of pestivirus was found in one CD patient, while one patient and one control were positive for CMV. No HSV 1 or 2, HHV 8 or enteroviruses were found. The serologic tests revealed that five of seven CD patients had antibodies against the early protein, the capsid protein and the EBV nuclear antigen (EBNA). The titers were not significantly altered post-surgically. None of the patients had antibodies of the IgM isotype. Our findings vary from those of Ruther et al. who demonstrated evidence of EBV in tissues from 7 of 11 (64%) German CD patients. Antibodies to early EBV viral antigen and to nuclear antigen in five of seven Belgian patients suggest persistent active viral infection.

The activation of the IFNß induction/signaling pathway in porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus is variable.

BACKGROUND: It has been recognized that the expression of type I interferon (IFNα/ß) may be suppressed during infection with porcine reproductive, respiratory syndrome virus (PRRSV). This causes profound negative effects on both the innate and adaptive immunity of the host resulting in persistence of infection. OBJECTIVE: Test the effects of PRRSV infection of porcine alveolar macrophages (PAMs), the main target cell, on the expression of interferon beta (IFNß) and downstream signaling events. METHODS: In order to examine those effects, PAMs harvested from lungs of healthy PRRSV-free animals were infected with virulent, attenuated, infectious clone-derived chimeric viruses, or field PRRS virus strains. Culture supernatants from the infected PAMs were tested for IFNß protein expression by means of indirect ELISA and for bioactivity by a vesicular stomatitis virus plaque reduction assay. The expression of the Mx protein was assayed to ascertain signaling events. RESULTS: These experiments demonstrated that PRRSV does induce variably, the expression of bioactive IFNß protein in the natural host cell. To further elucidate the effects of PRRSV infection on IFNß signaling, Mx-1 an interferon stimulated gene (ISG), was also tested for expression. Interestingly, Mx-1 expression by infected PAMs generally correlated with IFNß production. CONCLUSION: The results of this study demonstrate that the induction of IFNß and signaling in PAMs after PRRSV infection is variable.

ELISA Serology for Antibodies Against Chlamydia trachomatis in Crohn's Disease.

BACKGROUND: Recently we reported IgA anti-Chlamydia antibodies in patients with Crohn's disease (CD), in particular in four patients from a single family of six with CD. METHODS: We studied sera from four cohorts from the north of France. These were identified as: EPIMAD (80 pediatric onset CD and 20 pediatric onset ulcerative colitis), MINOTOR (148 adult onset sporadic CD and 50 adult onset ulcerative colitis), Grande Famillies (50) and matched controls for the Grande Famillies cohort (49). Sera were tested using commercial anti-Chlamydia trachomatis (LGV2:434) IgG and IgA human enzyme-linked immunosorbent assay (ELISA) kits. Cutoff for positivity was 11.0 standard units. RESULTS: Patients with sporadic CD, unaffected first degree relatives from multiplex families and ulcerative colitis patients had no greater serologic reactivity than controls. However, multiplex families' patients had twice as many positives as the other groups: for IgG 20% vs. 8%; for IgA 20% vs. 10%. CONCLUSIONS: Though not attaining statistical significance, the data showed that familial CD patients had greater exposure to C. trachomatis than sporadic CD patients, supporting our earlier results from one family from the north of France. More specific serologic tests based on outer membrane proteins will need to be employed against the various Chlamydia species with zoonotic potential.

Molecular characterization of rotavirus isolated from alpaca (Vicugna pacos) crias with diarrhea in the Andean Region of Cusco, Peru.

Alpacas (Vicugna pacos), a species of South American camelids (SAC), suffer high morbidity and mortality from infectious diseases. Diarrhea is one of the leading causes of alpaca cria mortality in Peru and elsewhere. In order to develop appropriate control and/or treatment, it is necessary to identify infectious pathogens that cause diarrhea in crias. Rotavirus was isolated in cell culture from feces collected from crias with acute diarrhea that tested positive to rotaviral antigen by rapid immunochromatographic methods in an earlier study. The isolates were identified as rotaviruses by RT-PCR run with specific primers for human rotavirus VP7 coding sequences using total RNA extracted from cells displaying cytopathic effects as template. These alpaca isolates were further identified as group A rotaviruses by means of a VP6-specific PCR and were designated as ALRVA-K'ayra/Perú/3368-10 and ALRVA-K'ayra/Perú/3386-10. Molecular G and P typing, placed the former as G3/P11 and the latter as G3/P?. Sequence analysis of two genome segments (coding for VP4 and VP7) from the alpaca isolates revealed partial homologies to swine and human rotaviruses, respectively. These results demonstrate that rotaviruses are associated with a proportion of cases of diarrhea in crias, although prevalence and impact remain to be determined. The isolation of rotaviruses from alpaca crias with diarrhea will contribute positively to further understand the pathogen and its role in the diarrhea complex.

A Novel Vaccine Using Nanoparticle Platform to Present Immunogenic M2e against Avian Influenza Infection.

Using peptide nanoparticle technology, we have designed two novel vaccine constructs representing M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with Mono-M2e or Tetra-M2e with and without an adjuvant. Two weeks after the second boost, chickens were challenged with 107.2 EID50 of H5N2 low pathogenicity avian influenza (LPAI) virus. M2e-specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased M2e-specific IgG responses for each of the constructs as compared to a non-vaccinated group. However, the vaccine construct Tetra-M2e elicited a significantly higher antibody response when it was used with an adjuvant. On the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. The level of protection was evaluated using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. The Tetra-M2e with adjuvant offered statistically significant (P < 0.05) protection against subtype H5N2 LPAI by reduction of the AI virus shedding. The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against AI.

Pregnancy increases the risk of mortality in West Nile virus-infected mice.

West Nile fever outbreaks in the USA have caused over 700 human deaths, primarily due to neurological disease. The usual transmission route of West Nile virus (WNV) involves mosquito bites; however, alternative routes, including intrauterine infection, have also been reported. Here, the pathogenicity of WNV in mice during gestation has been investigated. An extremely high mortality rate was observed in pregnant mice (98 %, 60/61) compared with non-pregnant mice (52 %, 28/53; P<0.001), independent of the infecting dose or the week of pregnancy. Antibody titres were similar between pregnant and non-pregnant mice and between surviving and non-surviving animals. WNV RNA titres in brains were also similar between pregnant and non-pregnant mice. WNV RNA could be detected in placentas and fetuses. These observations suggest strongly that, in the mouse model, pregnancy increases the risk of severe WNV infection and may help to understand the pathogenic mechanisms involved in WNV infection during pregnancy.